To determine the chemical basis for specificity of tRNA in accepting amino acids and the mechanism by which aminoacyl-tRNA synthetases act, analyses of reactions of specifically modified tRNA and enzymes will be carried out. Reactions that modify individual bases will be carried out, active and inactive forms of tRNA will be separated and the sites of modified bases will be determined as has already been done in the modification of cytosine by bisulfite. Chemical cleavage of tRNA by base-specific reagents will also be carried out to isolate fragments for physical and biological characterization. Enzymatic cleavage that results in inactivation of tRNA will be carried out with limiting amounts of nuclease to determine the kinetics of hydrolysis at various positions and the relationship of specific cleavages to biological activity. Chemical modifications of specific amino acid residues in synthetases will be carried out and the modified enzymes will be examined for altered binding of tRNA as well as altered catalytic activity. These studies will be caried out primarily with arginine and lysine activating enzymes of E. coli and their cognate tRNAs.